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Article

Characteristics of Fragaria vesca Yield Parameters and Anthocyanin Accumulation under Water Deficit Stress

• Rytis Rugienius ^1,
• *
• , Vidmantas Bendokas ¹ , Tadeusas Siksnianas ¹, Vidmantas Stanys ¹, Audrius Sasnauskas ¹

and Vaiva Kazanaviciute ²

1

Lithuanian Research Centre for Agriculture and Forestry, Department of Orchard Plant Genetics and Biotechnology, Institute of Horticulture, LT-54333 Babtai, Lithuania; [email protected] (V.B.); [email protected] (T.S.); [email protected] (V.S.); [email protected] (A.S.) Department of Eukaryote Gene Engineering, Institute of Biotechnology, Vilnius University, LT-10257 Vilnius, Lithuania; [email protected]

2

*

Correspondence: [email protected]; Tel.: +370-37-555253

Abstract: Plants exposed to drought stress conditions often increase the synthesis of anthocyanins—natural

plant pigments and antioxidants. However, water deficit (WD) often causes significant yield loss. The aim of our study was to evaluate the productivity as well as the anthocyanin content and

composition of berries from cultivated Fragaria vesca “Rojan” and hybrid No. 17 plants (seedlings)

grown under WD. The plants were grown in an unheated greenhouse and fully irrigated (control)

or irrigated at 50% and 25%. The number of berries per plant and the berry weight were evaluated

every 4 days. The anthocyanin content and composition of berries were evaluated with the same

periodicity using HPLC. The effect of WD on the yield parameters of two evaluated F. vesca genotypes

differed depending on the harvest time. The cumulative yield of plants under WD was not less

than that of the control plants for 20–24 days after the start of the experiment. Additionally, berries

accumulated 36–56% (1.5–2.3 times, depending on the harvest time) more anthocyanins compared

with fully irrigated plants. Our data show that slight or moderate WD at a stable air temperature of

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Citation: Rugienius, R.; Bendokas, V.; Siksnianas, T.; Stanys, V.; Sasnauskas, A.; Kazanaviciute, V. Characteristics of Fragaria vesca Yield Parameters and Anthocyanin Accumulation under Water Deficit Stress. Plants 2021, 10, 557. https://doi.org/10.3390/ plants10030557

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about 20 C positively affected the biosynthesis of anthocyanins and the yield of F. vesca berries. Keywords: anthocyanins; deficit irrigation; drought; pigments; temperature

Academic Editors: Alfredo Guéra and Francisco Gasulla Vidal

1. Introduction

Abiotic stresses, including drought, have detrimental effects on the yield of various crops in different parts of the world, and agriculture in many regions has become uncompetitive as a result of this yield instability. Predicted climate changes in the near

future are expected to cause major challenges in modern agriculture due to their significant

potential negative impacts on both the quantity and quality of various crops, including

Received: 19 February 2021 Accepted: 12 March 2021 Published: 16 March 2021

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in published maps and institutional affiliations.

• strawberry [
• 1,2]. Water deficit is generally associated with a reduction in the size and yield

of strawberry fruits. However, some studies have demonstrated that water deficit might

increase the dry matter content, improve the concentration of health-related compounds,

and improve the taste of strawberry fruits [3–7]. Declining water resources are leading to

concerns about high water usage for some horticultural purposes. Studies to determine the

minimum necessary amount of water for crop development are required not only to save

water and/or energy, but also to improve plant fitness to resist biotic and abiotic stresses,

as well as improve nutritional, functional, and sensorial food properties [5,8].

Plants exposed to stress often increase the biosynthesis of phenolic compounds, particularly flavonoids, such as anthocyanins. As one of the most ubiquitous classes of flavonoids in berries, anthocyanins possess a multitude of biological roles, including protection against

solar exposure, and ultraviolet radiation, free radical scavenging and anti-oxidative capac-

ity, defense against many different pathogens, attraction of predators for seed dispersal,

Copyright:

• ©
• 2021 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and the modulation of signaling cascades [9]. Epidemiological and clinical studies suggest

• Plants 2021, 10, 557. https://doi.org/10.3390/plants10030557
• https://www.mdpi.com/journal/plants

Plants 2021, 10, 557

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that an anthocyanin-enriched diet may lower levels of certain oxidative stress biomarkers

in humans, and this could be associated with a reduced risk of cognitive decline and the

development of neurodegenerative and cardiovascular diseases, in addition to sustaining

hepatic function and kidney protecting activities [10]. Berry anthocyanins improve neuronal and cognitive brain functions and ocular health as well as protect genomic DNA

integrity. Anthocyanins assist in antiplatelet aggregation, act as an antiangiogenic barrier,

and display anticancer properties [11,12].

Anthocyanin composition, as well as that of other flavonoids, has been studied exten-

sively in wild and cultivated strawberry [13–18].This notwithstanding, the relation between

water deficit (WD) and anthocyanin accumulation is not fully understood, especially in fruit crops, and the potential to increase food value and reduce cultivation costs is not

fully exploited. Wild strawberry (Fragaria vesca) is a good model plant for various studies

because of its short cycle, small genome, and ease of reproduction [19]. Wild strawberry is

a relative of the cultivated strawberry Fragaria

and greenhouses. The aim of our study was to evaluate the growth and fruiting of F. vesca

×

ananassa, which is grown in open fields

plants and their anthocyanin content under water deficit in a greenhouse.

2. Results

2.1. F. vesca Yield Parameters

The number and yield of berries from fully irrigated control (I100) plants of both
“Rojan” and hybrid No. 17 remained relatively stable during the first two weeks of the

treatment in April (Figure 1a,b,e,f). Later, starting from the fourth–fifth pick, or 20–25 days

after the beginning of harvesting, the number and yield of berries started to increase. Berry ripening dynamics were affected by extreme air temperature, which increased to

24 ^◦C outsi_◦de starting on 8 May 2013. The temperature inside the greenhouse sometimes

reached 35 C. The rise in temperature accelerated berry ripening and led to increases in the

number and yield of ripened berries. This increase was variable and reached a maximum

on 20–24 May. The berry size decreased gradually until 8 May. Then, a temporary increase

in berry size, especially for hybrid No. 17, was observed (Figure 1c,d). This was also clearly

a consequence of the increased air temperature.

The impact of water deficit on yield parameters was genotype dependent. This impact was much more noticeable in hybrid No. 17 than in “Rojan”. The cumulative berry number

of hybrid No. 17 plants grown under water deficit was higher than that of fully irrigated

plants for almost the entire treatment period (Figure 1b). For 8 days of the experiment,

the cumulative yield of 50% irrigated plants (I50) did not differ from that of control (fully

irrigated) plants, and their yield subsequently became significantly higher and was the

highest at the end of the experiment compared with 25% irrigated (I25) and control plants

(Figure 1f). The cumulative yield of I25 plants was higher than that of I100 plants until

16 May, which is about two-thirds of the duration of the treatment. During the last 4 days,

it became the lowest compared with I50 and control plants. Interestingly, although water

deficit significantly (except for 8 May) decreased the average weight of the fruit of this hybrid, for most of the harvesting period, the cumulative yield was higher than that of

I100 plants (Figure 1d).

The cumulative berry number of “Rojan” plants grown under water deficit was also

higher than that of control plants for almost the entire duration of the experiment. However, the difference was much smaller than that of No. 17 plants, and at the end of the experiment,

the number of berries was the same among all irrigation variants of “Rojan” (Figure 1a). The cumulative yield of I50 “Rojan” plants did not differ from that of I100 plants for the entire experimental period. The yield of I25 plants from the 8th to 24th day of the

experiment (30 April–20 May) was higher than that of the control, but starting on 24 May,

it decreased, and at the end of the experiment, it was the lowest compared with other irrigation variants (Figure 1e). The difference in average berry weight among different

treatments was also much lower in “Rojan” than in hybrid No. 17 (Figure 1c). The berry

weight of I50 plants did not differ from that of I100 plants for almost the entire experimental

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period. Starting on 16 May, the berry weight of I25 plants significantly decreased compared

with that of the control, and it was the lowest at the end of the experiment. This decrease,

possibly combined with the lack of an increase in the number of berries, resulted in I25

having the lowest yield compared with the control and I50 plants at the end of experiment.

• Figure 1. Cumulative berry number (
• a,

b), average berry weight (

c,d), and cumulative yield per plant (e,f) of alpine

• strawberry “Rojan” ( ) and hybrid No. 17 (b,d,f
• a

,

c

,

e

) in different picks. Plants were fully irrigated (I100) and irrigated with

half (I50) and a quarter (I25) of full irrigation. Data followed by the same letter are not significantly different according to

the least significant difference (LSD) test at p ≤ 0.05.

In hybrid No. 17 plants under half irrigation (I50), the total yield increase was 6.2%

compared with control plants. The decreases in total yield per plant of hybrid No. 17 and

“Rojan” I25 plants were 8.6% and 21.0%, respectively.

The experiment for the least irrigated (I25) plants was completed on 24 May, when

plants became dry, and they died before 28 May. Clear signs of wilting were visible in the middle of May.

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2.2. Anthocyanin Profile and Concentration in Berries during Harvesting

The anthocyanin concentration of fully irrigated F. vesca berries of both varieties varied

from 3.6 to 4.7 mg/100 g fresh weight and remained stable until 4 May (Figure 2a,c). Then,

the anthocyanin concentration decreased more than two-fold in both varieties until the

end of the harvesting season. It was observed in our study that WD caused a considerable

increase in anthocyanin content in wild strawberry fruits, especially at the beginning of the harvest. The berries of “Rojan” I50 and I25 plants accumulated 1.5 and 2.3 times more anthocyanins then fully irrigated plants after 4 days of the treatment (Figure 2a).

Anthocyanin concentrations in the berries of hybrid No. 17 were 1.5–1.6 times higher than those of fully irrigated plants under both deficit irrigation regimes (Figure 2c). Differences

in the total amount of berry anthocyanins obtained under full and deficit irrigation regimes

were significant (p

≤

0.05) for both cultivars. When analyzing the cumulative anthocyanin

content at the end of study, we observed clear differences between irrigation treatments starting on the eighth day of the treatment for “Rojan” and on the 12th day for hybrid

No. 17. At the end of the study, the cumulative amount of anthocyanins in berries of I25

both “Rojan” and No. 17 plants was 56% and 36% higher, respectively, than that of fully

irrigated control plants (Figure 2b,d).

Figure 2. Dynamics of total anthocyanin content (mg/100g fresh weight) in the berries of wild strawberry “Rojan” (

a) and

No. 17 ( ) and cumulative anthocyanin content ( ), respectively. Plants were fully irrigated (I100) and irrigated with half

• c
• b,d

(I50) and a quarter (I25) of full irrigation. Data followed by the same letter are not significantly different according to the

LSD test at p ≤ 0.05.

We analyzed the anthocyanin composition of F. vesca berries and established that

pelargonidin 3-O-glucoside (Pel3G) was the most abundant anthocyanin. In “Rojan” and

No. 17 berries, Pel3G accounted for 50.7%

respectively, followed by cyanidin-3-O-glucoside (C3G), which accounted for 29.2% ± 8.3%

and 27.4% 5.5% of all anthocyanins, respectively. The percentage of peonidin-3-O- glucoside was 8.4% 1.2% and 10.1% 1.7%, respectively. The concentrations of other unidentified minor anthocyanins were low and together accounted for less than 10% of

±

7.5% and 50.6%

±

4.4% of all anthocyanins,

±

• ±
• ±

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total anthocyanins. In examining the ratio of anthocyanins, we focused on the first two

anthocyanins, as their levels were the highest. A tendency of C3G to decrease and Pel3G

to increase during harvesting was clearly apparent, although the variation was slight

throughout the experiment. This tendency was more consistent in the berries of “Rojan”.

The Pel3G/C3G ratio in the berries of this cultivar was 1.31 at the start of the treatment

and was relatively stable (up to 12 days after the start of treatment), and then it increased

significantly: on the 28th day, it was 2.9 times higher compared with the beginning of the

study period (Figure 3a). In contrast to this variety, a different trend was established in

berries of hybrid No. 17: the Pel3G/C3G ratio decreased at the beginning and subsequently

began to increase, and it had increased by 1.4 times at the end of the study compared with

the start (Figure 3b). The proportion and dynamics of both main anthocyanins in berries

during harvesting did not depend on water deficit. However, the Pel3G/C3G ratio in the berries of No. 17 plants irrigated at 25% (I25) was up to 35% and 20% lower in the

beginning and at the end of the study compared with that in fully irrigated plants.

Figure 3. Pel3G/C3G ratio in the berries of wild strawberry “Rojan” (a) and No. 17 (b). Plants were fully irrigated (I100)

and irrigated with half (I50) and a quarter (I25) of full irrigation. Data followed by the same letter are not significantly

different according to the LSD test at p ≤ 0.05.

3. Discussion

Water deficit in strawberry fruits is generally associated with a reduction in berry

size and yield. Previous studies have shown that drought stress decreases yield and fruit

weight, as well as leaf area, leaf dry matter, shoot dry matter, total dry matter, relative

water content, and stomatal conductance [3,5,20,21]. Liu and co-authors [22] showed that

water deficit and partial root-zone drying decreased the berry weight of the strawberry

(“Honeoye”), but the berry number remained stable.

The impact of our WD treatments on different yield parameters (berry number and

berry weight) of wild strawberry depended on the variety. Insufficient irrigation resulted in decreased berry weights for strawberry hybrid No. 17 and, to a much lesser extent, “Rojan”,

but it increased the berry number and yield at the beginning of the treatment. WD stress

has been reported to stimulate both berry set and ripening speed [23]. Mild WD was found to increase apple yield [24]. An increase in berry yield per plant of a strawberry hybrid was

observed in our other experiment [25]. This indicates that the dehydration stress-induced

increase in strawberry yield, at least during certain growth stages, is not coincidental.

Differences in berry set in response to water stress between the evaluated varieties may be explained by the genetic background of hybrid No. 17. One of its parents is the woodland strawberry F. vesca, which has probably evolved with different adaptive

characteristics from those of the cultivated alpine strawberry.

Differences among genotypes have been observed by other authors investigating the

response of strawberry plants to WD [20–27]. The reaction to water deficit stress in plants

of different genotypes has been related to the ratio between carbon fixation and water

loss [20] or differences in the response to jasmonic acid and stomatal closure speed [28].

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The rise in temperature before 8 May further accelerated the ripening of berries in all

studied irrigation regimes. An increase in berry yield due to elevated air temperature was

also observed in our other experiment [25]. In both cases, berry weight and yield decreased

at the end of harvest, especially when plants were grown under WD.

In our opinion, the changes in yield parameters under water deficit conditions in our

treatment can be divided into three stages. In the first stage, the air temperature was low

and stable, and water deficit caused berry ripening to slightly accelerate. The decrease in

the weight of the berries was variable and depended on the variety. In the second stage,

when the air temperature rose and fluctuated, the ripening of the berries accelerated even

further, but the stress due to water deficit also increased. The berry size decreased more

rapidly under a larger water deficit. In the third stage, the negative effects of water deficit

stress, exacerbated by higher temperatures, revealed themselves. The size of the berries

decreased substantially, the number of berries did not increase, and the yield decreased.

Plants need to endure different abiotic stresses, and polyphenols (including antho-

cyanins) accumulate in response to these stresses, helping plants to acclimatize to unfavor-

able environments. Hence, the concentration of these metabolites in plant tissue is a good

indicator to predict the extent of abiotic stress tolerance in plants, which varies greatly

among plant species under an array of external factors [29].

As mentioned before, in our experiment, water deficit caused a considerable increase

in anthocyanin content in wild strawberry fruits, especially at the beginning of the harvest.

According to data from several studies, water stress induces the accumulation of antho-

cyanin compounds in the berries of strawberry plants [5,21,30]. Bordonaba and Terry [3]

showed that differences exist in the way that different strawberry cultivars respond to drought stress, resulting in different fruit compositions. Despite the detrimental effect that WD can have on berry size, reducing water irrigation by a quarter between flower

initiation and fruit harvest has been found to result in better water use efficiency, as well as enhanced fruit quality and taste in some cultivars [3]. Mild drought and salt stress resulted in an increased content of phenolics, anthocyanins, and L-ascorbic acid in strawberry fruits;

however, fruit yield was not affected [5].

According to the data from our study, increase in anthocyanin content in wild straw-

berry fruits under water deficit had two peaks: the first was at the beginning of the

treatment, and the second was after the middle of the treatment, from 8 May. We propose

that the first peak was caused by slight or moderate water deficit stress, and the second peak

was the result of air temperature changes, and an increase in sunny hours. It is known that

the accumulation of anthocyanins and other polyphenols in plants is influenced by many

factors, including light and temperature [29]. Interestingly, anthocyanin accumulation had